Abstract Whereas tuberculosis (TB) incidence is decreasing, disease due to relatives of Mycobacterium tuberculosis, a group of intrinsically resistant bacteria called ?Non-tuberculous (non-TB) Mycobacteria? or NTM, is increasing. These emerging NTM pathogens specifically threaten vulnerable groups, including cystic fibrosis patients and aging populations. Treatment of NTM disease can require years of chemotherapy with multiple antibiotics to achieve cure, if cure is achieved. The most problematic NTM pathogen is Mycobacterium abscessus (Mab). Mab lung disease is considered not curable and death rates can exceed 50%. It was thought that Mab infections are acquired exclusively from environmental sources. However, recently it was demonstrated that Mab became transmittable from human to human. There is an urgent medical need for new antibiotics that work against this ?super-bug?. De novo - i.e. new chemical entities / new target - drug discovery takes 10 years or more to bring new medicines to patients. An alternative approach to new antibiotics is ?repositioning? of exiting drugs. Several antibiotics show some activity against NTM, however, issues such as low potency and toxicity limit or preclude their clinical use. We refer to ?repositioning? as the pathogen-specific chemical optimization of antibiotic classes that act against pharmacologically validated targets, but have been developed for infectious diseases other than NTM. Since these drug classes include members that are FDA-approved, attrition rates are lower and the probability of success is higher than incurred through de novo drug discovery. For instance, the oxazolidinone linezolid (target ribosome) shows some anti-Mab activity but suffers from low potency and toxicity. In screens of oxazolidinone libraries and follow-up characterization work with Merck we have identified lead compounds with improved potency and reduced toxicity, thus validating the strategy. Similarly, the rifamycin rifampicin (target RNA polymerase) shows poor activity against Mab. We identified rifamycin lead compounds exhibiting improved potency. Here, we will subject our two leads to optimization campaigns to deliver preclinical development candidates with tolerability, exposure and efficacy in established and novel mouse models of Mab lung disease. NTM infection models do exist, however, they largely rely on immune-deficient mouse strains and a systemic infection approach, whereas natural transmission occurs by inhalation. Importantly, current models show limited similarities in pathology to human NTM lung disease. Robust, more predictive mouse models are needed. Standard mouse strains are mostly resistant against Mab infections and clear the bacterium. In preliminary work we tested a small set of clinical Mab isolates against different wild type mouse strains and could show that some combinations deliver improved bacterial growth and pathology. In parallel to our two lead optimization projects (aim 1 and 2), we will develop and utilize improved Mab mouse models (aim 3). In conclusion, our work will deliver preclinical development candidates for progression to clinical development for Mab lung disease and a more robust and predictive mouse model.